JP2023525333A - Magnetic cleavage of compounds - Google Patents

Abstract

The present invention provides a method for cleaving at least one covalent bond in at least one molecule, said method comprising: (a) colliding at least one magnetic substance with said at least one molecule; and/or (b) causing collisions between at least one non-magnetic particle and said at least one molecule by moving said at least one magnetic body. In embodiments, the molecule is in a suspension or solution, (a) the solution or suspension is a liquid sample such as a buffer or a body fluid, the molecule is a nucleic acid, (b) the solution or suspension is The turbidity is drinking water or pool water, the molecule is a macromolecular contaminant, (c) the solution or suspension is sewage, the molecule is a biomacromolecule, (d) the solution or the suspension is a food or beverage and the molecule is gluten; , conditioners or soaps, wherein the molecule is a protein, polypeptide and/or peptide; (f) the solution or suspension comprises a macromolecular toxin, the molecule is said macromolecular toxin, or (g) the solution or suspension contains an undesirable enzymatic activity and the molecule is a protein exhibiting said enzymatic activity.

Description

The present invention provides a method for cleaving at least one covalent bond in at least one molecule, said method comprising: (a) colliding at least one magnetic substance with said at least one molecule; and/or causing collisions between at least one non-magnetic particle and said at least one molecule by moving said at least one magnetic body.

A number of documents are cited in this specification, including patent applications and manufacturer's manuals. The disclosure of these documents, while not considered relevant for the patentability of this invention, is hereby incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated and incorporated by reference.

Many industrial processes require the cleavage of relatively large molecules. This is because, while these molecules have undesirable properties, their cleavage products have acceptable or even desirable properties. Examples of such relatively large molecules include molecules of biological origin ranging from proteinaceous molecules to carbohydrates and nucleic acids. An exemplary process is to remove undesired enzymatic activity by cleaving the enzyme that confers said activity, detoxify a composition comprising a toxic protein such that the cleavage products of the toxin no longer exhibit toxic activity. , manufacturing nucleic acid-free reagents such as buffers, particularly long nucleic acid-free buffers.

Enzymes are often used for these cleavage processes. However, these enzymes need to be produced or obtained in large quantities, considering that the aforementioned processes are routinely carried out on a large scale. Thus, enzyme production is not only a complex process, but can also carry the risk of contamination, for example when live cells are used for production or when enzymes have to be purified from animal sources. Also, although enzymes remain the first choice for the described cleavage processes in many cases, this is a further alternative of biological origin in order to reduce or eliminate undesired activities of biomolecules. It should be noted that this means that drugs are used. Also, enzymes are delicate substances and typically retain activity only under very strictly defined conditions, where modifications such as oxidation or denaturation by pH or heat cause loss of function. Sometimes.

The use of magnets is generally not incompatible with processing molecules and compositions of biological origin. A very popular use is the magnetic stir bar, which rotates under the influence of an external magnetic field, resulting in rapid mixing of liquids, solutions and suspensions.

US 2010/068781 discloses a container having a magnet inside, the magnet exactly matching the cross-section of the container so that only a thin liquid film exists between the surface of the magnet and the inner wall of the container. Are listed. Magnets cannot move freely.

US 6,806,050 describes a device for handling paramagnetic particles as commonly used in analytical procedures.

In view of the above drawbacks, there is a need for improved means and methods of cleaving molecules with undesired activity, said means and methods allowing the release of yet another chemically or biologically active agent. does not require use.

The technical problem underlying the present invention is therefore that of such means and methods, more particularly non-chemical and non-chemical methods of cleaving or fragmenting chain molecules and macromolecules, including those of biological origin. It can be seen to provide non-enzymatic means and methods.

This technical problem is solved by the subject matter of the claims and the aspects and embodiments disclosed below.

More specifically, in a first aspect, the invention provides a method of cleaving at least one covalent bond in at least one molecule, said method comprising: (a) at least one magnetic substance; and/or (b) causing collision between at least one non-magnetic particle and said at least one molecule by moving said at least one magnetic body. A method is provided, comprising:

The term "cleavage" refers to the breaking of covalent bonds such that fragments of the starting molecule are formed. Cleavage can directly result in stable fragments or intermediate reactive species, which react with other molecules present to produce stable products. Other molecules may include water or, more generally, any molecule capable of forming stable adducts with the reactive species to the extent they are formed. The terms "cleavage" and "fragmentation" are used interchangeably herein with the terms "cleavage product" and "fragment".

The cleaved covalent bond is not particularly limited. Includes single bonds, and double and triple bonds, all of which are between atoms of the same type (eg, C—C bonds) or between different atoms (eg, C—N or C—O bonds) obtain. Preferred are bonds that occur in functional groups that link macromolecular building blocks, such as peptide bonds, ester bonds including phosphoester bonds, and glycosidic bonds. Such preferred bonds generally have lower bond energies compared to e.g. It is understood to be susceptible to cleavage.

As will become apparent below, the present invention allows control of the amount of energy transferred to the material being processed, thereby providing a means of targeting covalent bonds according to their bond energies. do. That said, there is generally a wide range of energies to be transferred, resulting in simultaneous cleavage of different types of molecules over said range; see Examples.

The number of fragments obtained from a given molecule is not particularly limited and includes 2 fragments and any higher number such as 3, 4, 5, 6, 7, 8 , 9, 10, 20, 50, 100, 200, 500, 1000 or more fragments. Based on the non-enzymatic and non-chemical cleavage processes of the present invention, previously unattainable fragmentation patterns can be generated. In this context, the term "fragmentation pattern" refers to both the site of fragmentation and the average size and size distribution of fragments obtained from a given molecule.

The size of the obtained fragment is also not particularly limited. The size can preferably be controlled by parameters (frequency, amperage, power, flux density, etc.) that control the methods further disclosed below. For example, if only one, two, or one order of magnitude smaller parts are cleaved compared to the molecule, the size of the fragments may be 1, 2, 3, 4, 5, 6 It can range from 1, 7, 8, 9, 10, or any other double digit number of atoms to fragments comparable in size to the molecule.

The term "at least one molecule" includes applications that use exactly or approximately one molecule, also called "single-molecule applications." An amount of 1 fmol to 1 μmol is encompassed by the term "at least one molecule" when there is no need to examine or deal with individual molecules, but a small number of molecules need to be fragmented. Macroscopic amounts are also included, for example in the range of 1 μmol to 10 ⁶ mol. The latter application is also preferred in view of the preferred embodiments described in more detail below, including processes routinely practiced on a large scale in the areas of water, beverage, and food processing. Especially in the context of the application of the invention in flow-through reactors, there is practically no limit to the amount to be treated, which can be well above 10 ⁶ mol, eg 10 ⁷ -10 ¹⁰ mol. Indeed, in the context of such continuous processes (see further below for details) the absolute amount is not particularly limited. For example, flow rates from 1 mol per hour up to 10 ⁶ or 10 ⁷ mol per hour are envisioned.

It is understood that other molecules may and generally will be present in addition to the molecule to be cleaved when practicing the methods of the invention.

Furthermore, the molecules to be cleaved may belong to a single molecular species or form a family of more or less closely related molecules, eg polyclonal antibodies.

Mixtures of both types of molecules to be considered are envisioned: mixtures with molecules that should be cleaved, and molecules for which cleavage is unnecessary or should be avoided.

The term "collide" refers to relative motion between the magnetic material and the molecule. A collision can occur more than once. A collision will generally involve the magnetic body and the molecule coming into close contact. Under such conditions of spatial proximity, energy transfer between moving magnetic bodies (or moving non-magnetic particles; see below) is possible, imparting the necessary energy to the molecule for bond cleavage.

Alternatively or additionally, for example, if multiple magnetic bodies are used and/or if one or more non-magnetic particles (see further below for details) are present, the motion of a given magnetic body is the molecular can act as a trigger for collision with another magnetic or non-magnetic particle. In other words, a given magnetic body may collide with a given molecule and/or cause collisions of said molecule with other magnetic bodies or particles, both types of collisions leading to covalent bonding of said molecule. can cause cleavage of The latter type of collision is also referred to as an "indirect" effect of the movement of said magnetic body and is defined by item (b) of the method of the first aspect. Options (a) and (b) will generally occur together when at least one magnetic and at least one non-magnetic particle of the bead is present. However, fragmentation due to purely indirect effects is also envisioned.

In addition to cleaving covalent bonds, the methods of the invention can also be used to disrupt other types of intra- and intermolecular interactions such as van der Waals interactions, hydrogen bonding, and hydrophobic interactions. .

The method can be performed using a single magnet, ie, a single magnetic body that can be implemented as a piece of ferrimagnetic, ferromagnetic, or paramagnetic material. Alternative implementations of the magnetic material are further disclosed below.

Multiple magnetic bodies or magnets may be used instead of said single magnet or single magnetic body. The number of magnetic bodies is not particularly limited, but the number of magnetic bodies in one or two digits, for example, exactly 2, 3, 4, 5, 6, 7, 8 1, 9, 10, 15, 20, 25, 30, 40 or 50 magnetic bodies or magnets are preferred. The number of magnetic bodies is understood to be limited to the extent that this is specified. In other words, the method of the first aspect may include other means, but such possibilities do not extend to the option of having more magnetic material than is explicitly specified. The successful performance of methods using different numbers of magnets is demonstrated in the examples.

For magnetic bodies and magnets, ferromagnetic and ferrimagnetic materials are preferred.
Therefore, magnetic bodies and magnets, in their preferred implementations, should be distinguished from paramagnetic particles commonly used in analytical procedures. Furthermore, while paramagnetic particles are in the μm range, the magnets and magnetic bodies of the present invention are preferably in the mm to cm size range, and even larger for larger vessels, tubes, or reactors. good.

Generally, the method of the invention is practiced within a defined space, such as a reactor. Said space may also be defined by the total volume of a solution or suspension only if said at least one molecule is in solution or suspension.

Under such circumstances, the number of magnetic bodies can also be indirectly defined in terms of their total volume. Preferably, said total volume occupied by all magnetic bodies together is between 0.01% and 98% of the reactor volume, solution volume or suspension volume, more preferably 0.01% to 98%. is between 1% and 75%, 0.1% and 50%, 0.1% and 40%, 0.1% and 30%, 0.1% and 20%, or 0.1% and 10% .

The structure and origin of the molecules to be cleaved are not limited. Macromolecules of biological origin are preferred; see below for details. Embodiments demonstrate that the method according to selected parameter settings allows cleavage of different types of molecules, and that said parameter settings are capable of cleaving different types of molecules while still providing cleavage of different types of molecules at each altered parameter setting. It proves that change is possible.

The inventors have surprisingly discovered that rapidly moving magnets can cause fragmentation of macromolecules. It should be noted that such fragmentation occurs when there is no established chemical or biological agent known to cleave a given macromolecule. This unexpected possibility is also referred to herein as "magnetic-induced cleavage" or "magnetic-induced proteolysis" when applied to proteins.

This unprecedented discovery constitutes a preferred embodiment of the present invention and paves the way for a range of applications that are described in greater detail below. What these applications have in common is that they overcome serious deficiencies of the technologically established processes. To further illustrate, to date, undesirable activity derived from a first biomolecule is often reduced or eliminated by adding an additional molecule of biological origin to the reaction mixture containing the undesirable activity. It is Although such additional molecules of biological origin, e.g. enzymes, have the advantageous property of cleaving or catalyzing the cleavage of the first biological molecule, reducing or eliminating undesired activity, this can also present undesirable contaminants in the final product obtained. This is exacerbated by the fact that many enzymes are not 100% pure because of their method of manufacture and further contain components that can have deleterious or immunogenic properties.

Moreover, the production of enzymes is generally a complex process with considerable costs.
The present invention is not limited to the removal or inactivation of unwanted molecules or activities. Applications focused on the products of cleavage, e.g., if the resulting fragment or one of the fragments is used as an active agent (this term is not limited to drugs), pharmaceuticals, from a diagnostic point of view or Some applications are compounds of interest for analytical purposes.

The methods of the present invention each overcome these deficiencies or address such needs. There is no longer a requirement for chemical or biological agents to cleave macromolecules. This method is easy to implement. Exemplary or preferred instruments, devices and kits for that purpose are also subject of the invention and are further described below. Illustratively, this method was applied for the purpose of fragmenting nucleic acids; see Example 1. Application to proteins is similarly demonstrated in the examples.

In a preferred embodiment, said at least one magnetic body performs a fluctuating or oscillatory motion.

For example, the magnetic body moves up, down, left, and right, and this motion may have regular or repetitive components, but it need not be so, and the spatial direction is not particularly limited. Magnetic bodies can also rotate about one or more axes, usually in addition to translational motion. See further below for a more precise description of the types of movement envisioned. As long as a reactor, ie container, enclosure, or container is used, the magnetic material may, but need not, hit or repeatedly hit the walls of said reactor. Thus, although the magnetic body is constantly in motion (e.g., intermittent motion unless otherwise specified by a particular implementation of the method of the invention), such motion is generally not directed motion. . Also, although said motion may be random, it is generally centered around a mean position located within the mentioned enclosure or receptacle, and the magnetic body does not leave the reactor. Movement of magnetic bodies generally has one or more translational components, and the average position can be anywhere in the middle of the reactor. This movement is therefore different from the movement (rotation) performed by a magnetic stirrer, where the average position of the magnet is at or near the bottom of the vessel containing the liquid to be mixed or stirred.

The term "vibration" indicates regular motion, but the term "fluctuation" is broader and encompasses irregular motion as well. In that respect, there is no particular priority. Considering that in practice the magnet often collides not only with the molecule to be cleaved, but also with at least one wall of the reactor or enclosure used to house the magnetic material and the molecule. , irregular movements occur more frequently. In any case, so long as a plurality of molecules or macroscopic amounts thereof are to be treated by the method of the invention, the magnetic material is generally repeatedly repetitively associated with said macromolecules and all or substantially all to be cleaved. This motion ensures repeated contact with all molecules. As mentioned above, the movement of the magnetic material may also cause collisions of the molecules with further magnetic and/or non-magnetic particles to the extent they exist. In such cases, the action of a given magnetic body on covalent binding can also be referred to as 'indirect', as opposed to a 'direct' collision between said given magnetic body and the molecule. . Importantly, such indirect action is sufficient to cause cleavage, ie collisions between the magnetic body and the molecule are not required.

In other words, in a related aspect, the invention is a method of cleaving at least one covalent bond in at least one molecule, said method comprising: A method is provided comprising moving at least one magnetic body relative to.

See further below for preferred embodiments of non-magnetic beads. The size of said non-magnetic beads may be between the mass of the molecule to be cleaved and the mass of said at least one magnetic entity.

It is understood that said relative motion of said magnetic body to said molecule in the presence of non-magnetic beads causes collisions between said molecules and said non-magnetic particles. Of course, collisions between the magnetic material and the molecules, ie "direct collisions" can also occur in such a setting, but there is no requirement in that regard.

Preferably, said at least one magnetic body performs a oscillating motion or an oscillating motion.
In preferred embodiments of any of the previous disclosed aspects of the invention, said motion is caused by a varying or oscillating magnetic field.

Magnetic fields are a common means of controlling the position and/or motion of magnets. Since according to the invention it is taken into account that the magnetic body moves, in this preferred embodiment a varying or oscillating magnetic field is used. Any such magnetic field may be useful.

Preferably, said magnetic field is generated by an electric current. It is well established that electric and magnetic fields are interrelated, and in particular that electric currents produce magnetic fields. As a result, controlling the current is a means of controlling the magnetic field produced thereby.

Alternatively or additionally, said magnetic field may be generated or modulated by an external magnet, respectively. The term "external" means that such magnets are not located within the reactor. The external magnet can be a permanent magnet. When using an external magnet, the fluctuating or oscillatory movement of the magnetic body is caused by a corresponding movement of the external magnet relative to the magnetic body.

In preferred embodiments, the magnetic field is generated by an electromagnet. As used herein, the term "electromagnet", in its simplest implementation, encompasses that portion of an electrical conductor through which current flows when in use. For the purposes of better controlling the magnetic field or generating stronger magnetic fields, certain implementations of electromagnets are envisaged and are the subject of preferred embodiments disclosed further below.

In preferred embodiments, the current fluctuates or oscillates. This behavior may also be referred to as a general "wave". The amount of current is known as the amperage. The current temporal profile is also referred to herein as a "waveform".

In preferred embodiments, the amperage of said current as a function of time is (i) a rectangular function, (ii) a sine function, (iii) a trigonometric function, (iv) a sawtooth function, or (v) (i) to A combination or convolution of any one of (iv).

Considering that the current oscillates or fluctuates, this also applies to patterns (i)-(v), ie said rectangular and trigonometric functions are in fact repeating rectangular and trigonometric functions. there is The term "pattern" denotes a series of events in which a given basic event is repeated at least once. In a broader sense, the repetitions need not be exact repetitions, the length of rectangles etc. in the time graph may vary (this effectively means the frequency, preferred frequency, and frequency corresponding to the preferred time-dependent change of ).

All embodiments (i)-(v) are also referred to herein as "alternating".
Especially preferred are said rectangular functions (called rectangular waves or square waves), more particularly patterns of repeating rectangular functions. The inventors have surprisingly found that this pattern causes a particularly vigorous motion of the magnetic material, and such vigorous motion is particularly efficient with respect to cleavage. In said rectangular function, the time intervals of high current and low current (or off current) may be the same or different. That said, cleavage has also been demonstrated for several other temporal profiles of current; see Example 6.

Means for controlling the length of said time intervals are known to those skilled in the art, for example these are referred to as Pulse Width Modulation (PWM). It should be noted that the energy transferred to the reaction mixture is governed not only by the frequency and amplitude of the current, but also by the relative duration of said time intervals.

In a further preferred embodiment of the method of the first aspect, said collision transfers an amount of energy to said molecule sufficient to cleave at least one covalent bond. It is well established that collisions are a means of transferring energy from one object to another. The energy received by the receptor can be converted into internal energy of said body (here a molecule) causing its fragmentation.

The energies involved in covalent bonds are known, and these energies also define the energies required for cleavage, especially in the absence of a catalyst. Most covalent bond energies are in the range of 100-1200 kJ/mol, see for example Chemistry: Atoms First 2e, ISBN 978-1-947172-63-0. Some examples of covalent bond energies are C—C bonds with bond energies of 345 kJ/mol, C—N bonds of 290 kJ/mol, and C—O bonds of 350 kJ/mol.

The preferred embodiments disclosed above are a means of demonstrating the overall results of the particular implementations chosen. The energy transferred to the molecule to be cleaved must be such that at least one covalent bond within said molecule receives the amount of energy necessary for dissociation.

As a rough estimate, the amount of energy transferred to a covalent bond by a magnetic body is less than or equal to the energy transferred to the magnetic body by a magnetic field. The energy of the magnetic field per unit volume is defined by E _mag =1/2B ² /μ ₀ , see further below for definitions of B and μ ₀ . E _mag is also less than or equal to the energy of the current generating the magnetic field. The energy of the latter can be estimated as E _curr =UIt, where U is the voltage, I is the amperage of the current generating the magnetic field, and t is the time the current flows. In other words, controlling any of B, U, I, and t is a means of controlling the amount of energy transferred to covalent bonds by the magnetic material.

That said, there are other means of specifying a detailed implementation of the method of the first aspect. This involves specifying one or more of the parameters that can be controlled or measured more directly. These parameters include the frequency and amperage of the current, and may also include the dimensions of the reactor and coils, as long as they are used. Also, the strength of the magnetic field, preferably at the location of the magnetic material, is a means of quantitatively specifying implementation details. In all these cases, a parameter value or combination of parameter values that ensures that the previous requirement (transfer of sufficient energy to cleave the bond) is met is preferred. Below, preferred ranges for the parameters mentioned are specified.

In a preferred embodiment, said current fluctuates or oscillates at a given frequency, preferably between 0.1 Hz and 20 MHz, more preferably between 10 Hz and 2 kHz, even more preferably between 50 and 500 Hz or between 90 and 300 Hz or between 100 and 200 Hz. do. Exemplary or preferred frequencies also include 70, 120, 130, 160, and 240 Hz. A further preferred range is therefore 70-240 Hz. It is understood that these measures apply not only to sinusoidal currents, but also to all current profiles specified herein including, for example, repeating rectangular patterns. The term frequency can also be applied to fluctuations, i.e. to non-regular time-dependent behavior (e.g. regular time behavior, also referred to herein as "oscillation"), where the time scale of fluctuations is a means of characterizing In such cases, the term "frequency" is understood to refer to the average frequency of variation.

The examples included herein demonstrate that the method of the invention works over a range of frequencies.

At least for reactors with volumes in the single to double order of mL range, and for the wells of a standard 96-well plate, lower frequencies of 80-300 Hz work particularly well, while about It can be seen that significantly higher frequencies, such as 1000 Hz, induce vibration of the magnetic material, but may not cause a full range of motion covering a significant portion of the reactor volume. This does not mean that higher frequencies are not beneficial. These frequencies may also be used in combination with lower frequencies (see below).

In more general terms, the preferred frequency range is that the magnetic body not only oscillates or rotates, but also performs a translational movement that explores the entire volume or substantially the entire volume of the material to be treated in the method of the invention. It is the range to be sure. In implementations that utilize a reactor and the material to be processed is in solution or suspension, the volume is the total volume of the solution or suspension contained in the reactor. In other words, although the instructions are given above for reactors and 96-well plates with volumes in the single to double mL range, the frequency range may vary with much smaller volumes, much larger volumes, or special geometries. may be necessary to adapt to reactors with By way of example, for smaller volumes such as the wells of a high-density microtiter plate (e.g., a 1536-well plate), higher frequencies, such as about 1 kHz, are comparable to the motion seen in larger vessels at lower frequencies. It is expected that the motion of the magnetic material will occur. In either case, the person skilled in the art, with the guidance given herein, can explore and optimize the parameters controlling the motion of said at least one magnetic body in a straightforward manner. Magnetic bodies that perform translational motion, preferably in addition to rotation, are preferred, as described further below.

In a further preferred embodiment said method comprises increasing the temperature of said magnetic body. This is to some extent an effect inherent in the use of magnetic fields to induce motion of the magnetic bodies. This is also known as induction heating. The increase in temperature can occur primarily at the surface of the magnetic material (also referred to in the art as "skinning") or can affect the entire magnetic material. Frequency (see above for preferred ranges) is a means of controlling the extent to which the temperature rises and/or the extent to which this occurs predominantly in magnetic bodies. By way of example, if heating of the surface of a magnetic material is desired, frequencies in the range of 10 kHz to 20 kHz are preferred. This frequency range is preferably applied in addition to the frequencies preferred for fragmentation.

In a preferred embodiment, said frequency is kept constant throughout the duration of said method.

In another preferred embodiment, said frequency varies as a function of time.
In a further preferred embodiment more than one frequency is applied at a given time. In such cases, each frequency of such multiple frequencies may be selected from any of the preferred intervals given above. Particularly preferred in the case of two frequencies, the first frequency is between 50 Hz and 500 Hz and the second frequency is between 80 Hz and 20 MHz. In other words, this preferred embodiment provides superposition of multiple frequencies. Further preferred ranges for the first frequency are those previously disclosed. Preferred ranges for the second frequency are 100 Hz to 100 kHz, 100 Hz to 10 kHz, 100 Hz to 5 kHz, such as 100 Hz to 1 kHz.

More than one frequency includes 2, 3, 4, 5, 6, 7, 8, 9 and 10 different frequencies. Such multiple frequencies may be applied throughout instead of a single frequency, meaning that they are applied throughout the implementation of the method. Also, multiple frequencies or different frequencies may be applied at different time intervals within a longer period of time. Within the longer period of time and in addition to the time intervals in which more than one frequency is applied, one or more, for example 2, 3, 4, 5, 6, 7, 8 , 9 or 10 time intervals, where only one frequency is applied.

The inventors have surprisingly found that regimes in which more than one frequency is applied perform better in terms of yield. "Yield" indicates the relative amount of fragmented molecules compared to the total number of (non-fragmented) molecules at the start of the method.

In a further preferred embodiment, said frequencies are not constant over time, if more than one frequency is applied, preferably between two or more frequencies, preferably periodically. is switched to or gradually changed to

Exemplary regimes are (120 Hz-1000 Hz) _n , (200 Hz-1000 Hz) _n , or (100 Hz-800 Hz) _n , where n is an integer, such as between 2 and 1000, such as between 10 and 1000. An integer between 100 and specifying the number of times the frequency pattern in brackets is repeated.

The duration of the time interval at constant frequency and/or constant amperage is not particularly limited. Time intervals of 1 second to 1 day, eg 1 minute to 1 hour are envisioned.

In a further preferred embodiment, said current (a) has an amperage I between 20 mA and 100 A, preferably between 0.1 and ²⁰ A; /m, preferably between 10 and 10 ⁶ A/m, and/or (c) 1 second to 1 week, such as 1 minute to 24 hours, 10 minutes to 5 hours, or 15 It is applied for a period of time t from minutes to 4 hours. Exemplary or preferred values that similarly define the lower and upper limits in relation to the ranges above include 30 minutes, 1 hour, and 2 hours.

The method of the invention has been exemplified over a range of time periods; see the examples below.

As previously disclosed, the amperage as a function of time fluctuates on a time scale governed by the frequencies disclosed herein, and that there is no constant amperage on the time scale of fluctuation. Please note. However, for practical purposes and in line with convention in electrodynamics, alternating current can be quantified in terms of its average amperage. The preceding values are average amperages in that sense. Note that the average preferably spans a timescale of variation. That is, so long as intermittent current is used, the average amperage is preferably within the ranges specified above when the current is on, and zero amperage when the current is off.

Magnetic field strength H determines the strength of the electric field and is measured in A per meter. H should be distinguished from the flux density B, which is particularly relevant in settings where the core is used to enhance the magnetic field of the current; see below.

In preferred embodiments, the amplitude of the fluctuations or oscillations is (a) constant or (b) varies over time, preferably on a slower timescale than the timescale of said fluctuations or oscillations.

This embodiment relates to the amplitude of motion of said current. The amplitude of the current oscillations or fluctuations is governed by the amperage.

In a further preferred embodiment said current is intermittent and/or said amperage changes over time, preferably periodically. This change over time is generally on a slower timescale than that defined by the frequency of the alternating current. In other words, if the alternating current is time-varying in the sense of this embodiment, the time dependence of the current is the superposition of two patterns or waves: the typically fast fluctuations inherent in alternating current, and the general Slower changes than normal.

An exemplary intermittent pattern is a repeating on (1 minute)-off (1 minute) sequence. Other preferred time intervals are given above. The advantage of the intermittent pattern allows the temperature to be kept constant or substantially constant, especially when heating of the reactor contents is observed.

In a further preferred embodiment a power of 0-1000W, preferably 1-200W is applied.

In a further preferred embodiment the current is supplied by a power supply. Preferably, the power supply has a potential or voltage U in the range of about 0-240V, eg 1-75V. These values refer to the average voltage applied.

In a further preferred embodiment, said electromagnet comprises at least one coil, preferably said coil comprises (a) a plurality of turns, such as between 1 and 10 ⁴ turns, preferably between 10 and 1000 turns; and/or (b) includes at least one Helmholtz coil, and/or (c) includes at least one core.

Examples of numbers of multiple coils are 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 150, 200, 300, 400, and 500.

The term "Helmholtz coil" is well-established in the art and refers to an arrangement of typically two identical coils spaced so that their axes of rotational symmetry are aligned or coincident. The magnetic field in the space between the coils is particularly homogeneous and/or particularly strong. The configuration of the at least one Helmholtz coil can be two Helmholtz coils.

A further configuration of multiple coils is known to result in an extended spatial region of particularly homogeneous magnetic field. A further example is the Maxwell coil.

The core serves to enhance the effect of the magnetic field. The core is preferably made of a ferromagnetic material such as iron, especially soft iron. The magnetic flux density B is related to the magnetic field strength as follows: B=μ _r μ ₀ H. μ ₀ is the magnetic permeability of a vacuum and is therefore a fundamental physical constant. _μr , on the other hand, is the relative permeability and determines the degree of enhancement of flux density by a given material, such as a ferromagnetic core, when under the influence of a magnetic field. Typically μ _r for ferromagnetic materials to be used as cores is between 10 ³ and 10 ⁶ , eg between 200,000 and 400,000, eg about 300,000. Suitable core materials include powdered metals, laminated metals, annealed metals such as annealed iron, ceramics, and solid metals.

Preferred values for B in the absence of core are between 10 ⁻⁸ and 10 ⁴ Tesla (T), such as between 10 ⁻⁵ and 1 T, such as single digit mT ranges, such as between 1 mT and 10 mT. between, or between 1 mT and 5 mT. If a core with a specific relative permeability is used, the value of B is multiplied by said relative permeability. Preferred values for B in the presence of the core are therefore between 10 ⁻² and 10 ⁹ , eg between 1 and 10 ⁶ T. Generally, these values refer to B at the site of a magnetic body or within the reactor.

As regards shape, the preferred coil is circular. Preferred diameters are between 1 mm and 1 m, or between 1 mm and 0.5 m, for example between 2 mm and 300 mm, or between 2 mm and 200 mm. Different shapes are also envisioned, such as coils having square, rectangular, or triangular shapes (ie, all or part of the windings are square, rectangular, or triangular). Finally, noting that coils are not a requirement for electromagnets, two anti-parallel wire configurations can also be used (“anti-parallel” means two wires (refers to the direction of current flowing through the

In a further preferred embodiment, more than 1 coil, preferably 2 to 10 ⁴ , such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 coils, or 10-1000 coils are used. It is understood that each coil may have one or more turns, the preferred number of turns being disclosed earlier herein.

In a further preferred embodiment, said magnetic body is (a) a single magnet, or (b) a collection of particles, at least one of which is a magnet, and said collection of particles comprises A collection of particles mediated by the magnetic field of said at least one magnet.

In its simplest configuration, said at least one magnetic body is realized by a single magnet. The size of a single magnet can vary widely and can be chosen appropriately depending on the dimensions of the reactor or vessel to be used. See below for this relative size criterion (magnet size vs. container size). As long as other substances present in the reactor or reaction mixture can be taken into account when choosing the size of the magnetic material, such other substances may be the molecule(s) to be cleaved and/or the non-magnetic beads. In addition to containing additional components of the sample.

For the sake of completeness, exemplary values for useful magnet sizes herein are between 0.1 mm and 10 cm, for example 0.2 mm, including any of the following values and ranges defined thereby: Given between ˜2 cm: 0.3 cm, 0.4 cm, 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, and 1 cm. The same preferred sizes and size ranges apply to any magnetic bodies considered herein, and also to implementations in which multiple magnetic bodies and/or magnets are used. Generally, these lengths refer to the maximum extension of the magnetic body. Thus, by way of example, a disk-shaped magnet may have dimensions such as 3×1 or 5×2 mm. Similarly, a cube can have dimensions between 1×1×1 mm and 5×5×5 mm.

Alternatively, multiple magnets as defined above may be used. Exemplary, yet non-limiting numbers are single and double digit ranges, eg, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Also, more than 10 magnets may be used, such as 20, 30, 40 or 50 magnets. These numbers refer to a single reactor or vessel even when a multiple vessel or reactor (eg, microtiter plate) configuration is used.

There are no particular restrictions on the shape of the magnet, and a shape that does not adversely hinder the free movement of the magnet is preferred. Exemplary shapes include sticks, bars, cylinders, rods, rods with rounded ends, cubes, cuboids, prisms, spheres, elongated and oblate ellipsoids, discs, tetrahedrons, octahedrons, dodecahedrons, and dihedrons. Includes decahedron.

The examples show that the method can be practiced with different numbers of magnets, as well as magnets of different shapes, sizes and materials.

Such setups still need to be distinguished from the previously disclosed "aggregates". The latter is where a large number of particles (in the simplest case a single particle or bead, such as a ferromagnetic bead) is coupled with a single or a small number (e.g. tens) of magnets (optionally according to the invention, reinforced by a magnetic field). It refers to a set-up that assembles under the influence of a magnetic field into a single magnetic body that essentially behaves like a single magnet. For the sake of completeness, multiple magnetic bodies can also be used, each of which, or a portion thereof, can be obtained from the above collection. However, this is highly unlikely under many circumstances. In particular, placing more than one aggregate in the same reactor or vessel can lead to the formation of a single larger aggregate (which is also in the magnetic field), unless measures are taken to prevent this from happening. also behaves like a magnet).

In a further preferred embodiment, (a) said magnet comprises or consists of a ferromagnetic material or ferrimagnetic material, and (b) the particles of said aggregate as defined above comprise ferromagnetic material, ferrimagnetic material, comprising or consisting of a material selected from magnetic, paramagnetic and/or diamagnetic materials, preferably from ferromagnetic and ferrimagnetic materials, and/or (c) the magnet and/or The particles are preferably coated with (i) coatings that impart chemical stability, (ii) coatings that impart mechanical stability or hardness, (iii) coatings with catalysts, (iv) nucleic acids such as probes and primers. (v) coating with chelating agents such as IMAC, _TiO2 and _ZrO2 ; (vi) (1) reverse phase groups such as C18, C8, benzene; (2) HILIC groups such as hydroxyl groups; (4) anion exchange groups such as primary, secondary, tertiary, and quaternary amino groups; and (5) (1) to ( 4) coating with a chromatographic material, preferably selected from any combination of any one of (vii) globulin, in particular immunoglobulin, streptavidin, biotin, protein A, protein G; Coating with preferred ligand binding proteins and/or their cognate ligands, enzymes such as oxidoreductases, transferases, ligases such as polymerases, hydrolases such as proteases, peptidases, nucleases, saccharidases, lipases, lyases and isomerases and (viii) coated with a coating selected from any combination of (i)-(vii).

Suitable materials for said magnets include the following elements and their alloys: Neodymium-Iron, Neodymium-Iron-Boron (eg Nd ₂ Fe ₁₄ B), Cobalt, Gadolinium, Terbium, Dysprosium, Iron, Nickel, Oxides. iron, manganese-bismuth, manganese-antimony, manganese-arsenic, yttrium-iron oxide, chromium oxide, europium oxide, and samarium-cobalt. Particularly preferred materials are neodymium-iron and samarium-cobalt.

In less preferred embodiments, paramagnetic materials can also be used to achieve what are termed "magnets", especially if such paramagnetic materials have a high magnetic susceptibility.

(c) Suitable coatings according to (i) include polypropylene, polyethylene, polystyrene, parylene, titanium nitride, polyimides, chloropolymers, and fluoropolymers, preferably polytetrafluoroethylene (PTFE).

In a further preferred embodiment, said at least one molecule is in solution or suspension. In such cases, the molecules to be fragmented are dissolved or suspended in a liquid phase and the magnetic material is located within said liquid phase (a significant amount of the total time of the process is required to ensure cleavage). at a rate of ).

Liquid handling is facilitated by containers. Therefore, in a preferred embodiment, said at least one molecule and said at least one magnetic substance are located within a reactor. As used herein, the term "reactor" is used generically in which said at least one molecule and said at least one magnetic material are located and interact ("react ”) refers to an enclosure or container that can be Alternatively, the term is not particularly limited and includes containers, closed-bottom containers, containers with lids, containers with closed-bottom lids, fully closed or sealed containers, tubular elements, and It contains elements with at least two openings that allow continuous liquid flow through an accordingly designed reactor. The reactor may also be implemented as a microfluidic device, ie a miniaturized device, comprising one or more channels with openings, optionally with extensions or reservoirs and/or valves.

In a preferred embodiment, the process is carried out batchwise, for example by using a reactor that is a closed-bottom vessel.

Exemplary preferred containers are configured to hold volumes between 5 μL and 1 L, preferably 10 μL and 50 μL, more preferably 30 μL, 40 μL, 100 μL, 150 μL, 200 μL, 250 μL, 500 μL, It is configured to hold volumes of either 1 mL, 1.5 mL, 2 mL, 5 mL, 15 mL, and 50 mL.

In a further preferred embodiment, the method is carried out in a continuous manner, e.g. the solution or suspension is preferably (a) in a reactor having at least two openings, the at least one magnetic body and/or (b) motion of the magnetic material occurs while the solution or suspension is flowing.

Item (a) defines that the reactor is a tubular element having at least two openings. Cross-sections can vary widely depending on the particular application and the scale of the process being performed. Thus, cross-sections between 0.1 mm and 1 m, for example between 0.5 mm and 10 cm, as well as values outside these ranges are envisioned.

Regarding shape, tubular elements may be a simple and convenient implementation, but other shapes are also envisioned considering that they provide flow-through. Such reactors may, and generally will, include means for controlling or altering the flow-through of the solution or suspension containing the molecule to be cleaved. In other words, flow-through is an additional parameter in addition to the parameter controlling the magnetic body motion that ultimately governs the performance, degree of cleavage, and yield of the method of the present invention.

In a further preferred embodiment, (a) the movement of said magnetic body is relative to said reactor and/or (b) the average position of said at least one magnetic body is preferably relative to said movement. Without being significantly limited, preferably (i) based on said magnetic field and/or (ii) a filter, mesh, membrane or sponge retaining said magnetic body or a thin wire connecting said magnetic body to said reactor , filaments, wires or springs are substantially constant with respect to the reactor.

Item (a) suggests that the relative motion of the magnetic body and the molecule simultaneously, the relative motion of the magnetic body and the reactor, in the simplest case the same kind of motion (i.e. if we neglect the motion of the molecule with respect to the reactor) It simply states what it does.

Item (b) provides means for preventing magnetic material from jumping out of the reactor or being washed out of the flow-through reactor.

In a further preferred embodiment, the dimensions of said at least one magnetic body and said reactor are such that said at least one magnetic body is free or substantially free to move about its mean position.

This embodiment is thus distinguished from designs for limited motion where only a thin layer of liquid can pass between the magnet and the inner wall of the container, for example a cylindrical magnet in a cylindrical container. and the inner diameter of the container is only slightly larger than the (outer) diameter of the magnet.

As can be readily seen, the free motion requirement is met for magnets with a maximum extension that is small compared to the dimensions or cross-section of the reactor, vessel, or tube, e.g., a 3 mm magnet in a 1.5 mL vessel. It is "Small relative to the vessel" generally means that the largest dimension of the magnet is smaller than the smallest dimension of the reactor, vessel, or tube. In other words, the maximum dimension of the magnet should be adapted to pass through the smallest passageway or cross-section in said reactor, vessel or tubular element. Smaller than minimum dimension preferably means 3/4, 2/3, 1/2, 1/3, 1/4 or 10% of said minimum dimension or cross-section of said reactor, vessel or tube. do. Thus, the maximum dimension of the magnetic body or magnet is 3/4, 2/3, 1/2, 1/3, 1/4, or 10% or less of said minimum dimension or cross-section of said reactor, vessel, or tube can be

As is clear from the foregoing, the magnetic body and container are not such that only a thin liquid sample film separates the magnetic body and the inner wall of the container. To further illustrate, if there is a thin film, shear forces will be generated when the magnetic material moves. Note that the magnetic material must be properly contained within the container for shear forces to occur. Therefore, free motion of the magnetic body cannot occur in such a setting. Otherwise there will be no proper fit, no thin liquid film and no shear forces.

In preferred embodiments, the free or substantially free movement is about at least two, at least three, at least four, at least five, or preferably all six axes of translational and rotational motion. , or along them, preferably said free or substantially free movement comprises translation along at least two axes.

For a point-like object, there are three degrees of freedom of motion, namely translation in three independent directions across three-dimensional space. For extended objects, there are three additional degrees of freedom, which can be defined by three independent axes of rotation.

The previous example (3 mm magnet in a 1.5 mL container) is considered a free motion implementation along or about all six axes. In particular, this includes freedom of movement along at least two enumerated axes of translational motion, and as such is clearly distinguished from the restricted motion designs referred to. Given the teachings herein, one of ordinary skill in the art can readily determine the most appropriate parameters of the method of the first aspect to provide such motion; .

In a further preferred embodiment, said at least one magnetic body collides with at least one wall of said reactor. This is not a requirement, but it is not a drawback. Collisions can occur repeatedly. It should be noted that even such an embodiment prefers free motion, like a 3 mm magnet in a 1.5 mL vessel, repeatedly bumping against the wall as it moves the liquid phase contained therein.

In a further preferred embodiment, the internal diameter of said coil or said at least one coil is such that said reactor or a plurality of reactors, for example an array of reactors, can be arranged within said coil.

An exemplary implementation of this embodiment is a cylindrical vessel or a vessel with a cylindrical cross-section surrounded by a coil.

Additionally, configurations with at least one coil above, below, or adjacent to the reactor are also envisioned and functional (importantly, the magnetic field is generated at the magnet). There is also no requirement for the magnetic body to be located at the point of maximum magnetic field strength of the magnetic flux density.

Similarly, assuming the vessel has a major axis of rotational symmetry, said axis may be aligned or even coaxial with the axis of symmetry of the coil, although this need not be the case. Any angle between the two axes may be used.

In a further preferred embodiment, the reactor has a circular cross-section and the inner diameter of the coil or at least one coil is only slightly larger than the outer diameter of the reactor. Depending on the elements used and the precision with which they are manufactured, "slightly greater" may include, for example, 1.0001, 1.001, 1.01, or 1.1 times.

In a further preferred embodiment, at least part of the wall of said reactor is magnetically permeable. More preferably, the entire reactor is made of magnetically permeable material.

Suitable materials include plastics, polymers such as polypropylene, glass, and ceramics. Metals can also be used, keeping in mind the permeability requirements.

Furthermore, at least one wall of the reactor may be made of, for example, polypropylene, polyethylene, polystyrene, parylene, titanium nitride, copper, nickel, silver, chromium, epoxy resin, zinc, tin, Everlube, paying attention to magnetic permeability requirements. , silica, phosphates, rubbers, chloropolymers and/or fluoropolymers, preferably gold, titanium nitride and polytetrafluoroethylene.

In a further preferred embodiment, said method comprises (a) an ambient temperature such as -100 to 100 degrees Celsius, preferably about 20 degrees Celsius, and/or (b) a pressure between 0.1 to 10 bar, preferably about It is carried out at ambient pressure, such as 1 bar.

In other words, the invention can be conveniently practiced under ambient conditions. That said, the exact conditions may depend on, or be optimized according to, the molecule to be cleaved. Changing the temperature and/or pressure may lead to better performance, such as using a protective gas atmosphere.

In a further preferred embodiment, said at least one molecule is a macromolecule or chain molecule, said macromolecule or chain molecule comprising or consisting of at least two building blocks, preferably said Building blocks are amino acids, nucleotides, ribonucleotides, sugars, saccharides, phosphates, fatty acids, glycerides, or combinations of any of the foregoing.

In other words, the term "molecule" includes proteins, peptides, polypeptides, nucleic acids, saccharides, and lipids. Nucleic acids include oligonucleotides and polynucleotides and can be or include RNA and DNA. Lipids include triglycerides, phosphatidyl compounds, and sphingolipids. The molecule may be a combination of or include any of the above. As long as the molecule is proteinaceous and of biological origin, it may be a protein with post-translational modifications, for example said protein may be N- and/or O-glycosylated, phosphorylated and /or may be prenylated.

The terms "macromolecule" and "chain molecule" are used to indicate that the molecule under consideration can be thought of as being made up of multiple identical or related building blocks. The term "chain" further suggests a linear topology of the molecule, and as is well known in the field of structural biology, chain molecules of biological origin have a linear primary structure while defined It is recognized that secondary and tertiary structures can be assumed.

Molecules include polymers, polycondensates, and combinations thereof, with respect to the chemistry of the junctions between building blocks. Polycondensates are chain molecules formed from monomers, during which water is expelled, more specifically when bonds between two adjacent building blocks are formed. This applies, for example, to polypeptides when they are formed from free amino acids and when they are assembled in ribosomes within living cells.

The molecular weight is not particularly limited and the number of building blocks is also not particularly limited and can be as low as 2, 3, 4, 5, 6, 7, 8, or 9. More generally, the general range for the number of building blocks is 10-100,000, eg, 50-1,000. A preferred molecular weight range is 1 kDa to 10 MDa, with values outside these ranges also intentionally contemplated, for example, when cleaving chromosomes, the molecular weight range is about 2× for human chromosome 1, for example. Up to 10 ⁵ MDa.

In a further preferred embodiment a further means for cleaving at least one covalent bond of said at least one molecule is contacted with said at least one molecule, said further means preferably comprising an enzyme, a chemical and a / or a catalyst. Chemicals include technology-established chemicals such as acids (eg, HCl, HNO ₃ , H ₂ SO ₄ , and HIO ₄ ) and bases (eg, NaOH and KOH). Useful catalysts, in addition to enzymes, include the following non-enzymatic catalysts: sulfonated carbonaceous surfaces, metal oxides, H-type zeolites, metal ions such as Zn2 ⁺ and Mg2 ⁺ .

While recognizing that it is accomplished by the present invention to completely avoid the means enumerated in this embodiment, for tailored or enhanced applications, the newly discovered "magnetically induced cleavage" and techniques It is believed that combination with well-established cleaving means may be beneficial.

In a more preferred embodiment, said enzyme is a protease if said molecule is a polypeptide, protein or peptide, a nuclease if said molecule is a nucleic acid or an oligonucleotide, and said molecule is a ribonucleic acid or an oligonucleotide. If it is a ribonucleotide, it is a ribonuclease; if said molecule is an oligo- or polysaccharide, it is a saccharidase or a glycosidase. Preferred proteases include trypsin, LysC, GluC, AspN, ArgC, and chymotrypsin.

It should be noted that the molecule can contain any combination of amino acids, nucleotides and sugars. For example, it may contain both a peptidic or proteinaceous moiety and a saccharide attached to said moiety, such as by a glycosidic bond. Examples are glycosylated proteins. Fragmentation of glycosylated proteins may comprise or consist of removal or fragmentation of saccharides and/or fragmentation of peptidic or proteinaceous portions. Glycosidases, among others, may be used as long as the fragmentation is improved or controlled by the additional means previously disclosed.

In another preferred embodiment, which may or may not be combined with the above additional cleaving means, non-magnetic beads may be added to the reaction mixture, eg the solution or suspension. Such non-magnetic beads are different from particles that are optionally contained in a magnetic body, and are also different from paramagnetic beads commonly used in assays. Non-magnetic beads can be ceramic beads, polymeric beads, glass beads, or metal beads, and the metal is a non-magnetic metal. As regards size, they preferably range between 1 μm and 5 mm, for example between 0.1 mm and 2 mm. Also, the non-magnetic particles preferably have the same or similar size range compared to the size of the magnetic body or magnet.

While not wishing to be bound by any particular theory, such beads can exert shear forces as they move about by the action of at least one magnetic material, which shear forces are then applied to said at least Facilitates the cleavage of a single covalent bond. In addition, it is believed that the non-magnetic particles limit the movement of molecules to be cleaved, increasing the probability of collisions between the molecules and the magnetic material (in other words, the number of collisions per unit time). This increases the yield or performance of the method of the invention.

In a further preferred embodiment, inert viscous liquids and/or gels such as polyacrylamide gels or agarose gels are added to said sample. Without wishing to be bound by any particular theory, it is believed that embedding the molecule(s) to be fragmented in a gel or viscous liquid can be obtained from a magnetic material or from any non-magnetic particles, if present. It is believed to be a means of improving energy transfer to the (s), thereby improving yield. In Example 1, agarose is used.

In a further preferred embodiment of the method of the first aspect of the invention, (a) the solution or suspension is a liquid sample such as a buffer or a body fluid, the molecule is a nucleic acid, and (b) the solution or suspension is The turbidity is drinking water or pool water, the molecules are macromolecular contaminants such as toxins, and (c) the solution or suspension is sewage, the molecules are chemicals such as antibiotics or peptide hormones. is a biomolecule, (d) the solution or suspension is a food or beverage, the molecule is gluten, and (e) the solution or suspension is a protein, polypeptide and/or peptide, for growth media and/or for personal care products such as shampoos, conditioners or soaps, wherein the molecules are proteins, polypeptides and/or peptides, and (f) a solution or suspension comprises a macromolecular toxin and the molecule is said macromolecular toxin; or (g) the solution or suspension comprises an undesirable enzymatic activity and the molecule is a protein exhibiting said enzymatic activity.

These preferred embodiments demonstrate how large-scale processes are likely to be improved by the methods of the present invention.

In particular, in those technically established implementations, processes for reducing nucleic acid content generally require UV light or filtering. This can be avoided by the process of the present invention. A reduction in nucleic acid content is understood to refer to long nucleic acids, especially those containing coding sequences. Fragmentation of such long nucleic acids by the method of the present invention yields shorter fragments, and by fine-tuning the method the average length of the fragments can be controlled. In contrast to long nucleic acids, fragments are generally tolerated in compositions such as buffers. Nucleic acids include DNA and RNA.

An exemplary field where nucleic acid contamination of reagents and buffers is undesirable is forensics. Example 1 demonstrates DNA fragmentation.

Providing drinking water and pool water that meets established standards of hygiene and health safety typically requires the use of aggressive chemicals such as chlorine or ozone. The present invention makes it possible to eliminate the handling of such hazardous materials.

Many toxins, especially highly toxic compounds such as botulinum toxin, diphtheria toxin, cholera toxin, and ricin, are of biological origin and are proteinaceous in nature. Fragmentation of these toxins by "magnetic-induced proteolysis" according to the present invention is a convenient means of controlling the risks arising from their potential presence in materials or compositions to be brought into contact with humans. For proof of principle, see Example 4, which shows that the activity of the enzyme is lost when treated according to the present invention.

The following further aspects are related thereto:
In a second aspect, the invention provides a method of reducing nucleic acid content in a buffer, said method comprising the method of the previously disclosed first aspect, wherein said buffer comprises said A method is provided that is a solution or suspension. Preferably, said nucleic acid is a long nucleic acid, ie a nucleic acid comprising one or more coding sequences.

In a third aspect, the invention provides a method of reducing unwanted enzymatic activity present in a solution or suspension, said method comprising: applying to said solution or suspension containing said enzymatic activity. See Example 4.

In a fourth aspect, the invention provides a method of detoxifying or sterilizing a solution or suspension comprising a macromolecular toxin, said method comprising: A method is provided comprising applying to a suspension.

Preferably, said methods of the second, third and fourth aspects are performed in a continuous manner as further defined above.

In a fifth aspect, the invention provides use of magnetic materials and means for generating a varying or oscillating magnetic field for cleaving at least one covalent bond in at least one molecule.

Preferred embodiments of the method of the first aspect apply mutatis mutandis to the use of the fifth aspect.

In a sixth aspect, the present invention provides (a) an electrical conductor, preferably at least one coil, more preferably a Helmholtz coil, (b) a vessel and/or array of vessels, such as a microtiter plate, and (c) comprising or consisting of at least one magnetic body in the container, or at least one magnetic body per container in the container array;
Said at least one magnetic body is produced in use by said electrical conductor, preferably by implementing said electrical conductor as a coil and by said container or said container array being located within said coil A device is provided in which elements (a) and (b) are configured to be under the influence of a magnetic field.

Such devices are adapted for the method of the first aspect. Note that there is no requirement that a separate coil be placed in close proximity to each vessel or array of vessels, or that each said vessel be surrounded by its own coil. Alternatively, a single coil of suitable dimensions can be used to accommodate the vessel array. Preferred in this context are the previously disclosed Helmholtz coils that produce a uniform magnetic field over an extended spatial region.

Preferred embodiments of the method of the first aspect apply mutatis mutandis to the device of the sixth aspect, so long as they provide details of the electrical conductor, container(s) and magnetic body. be.

In a preferred embodiment of the device of the sixth aspect, said device further comprises or further consists of a control unit, said control unit being adapted to implement the method of the first aspect.

In a particularly preferred embodiment, said control unit has a loaded computer program product, said computer program product comprising instructions for implementing the method of the first aspect.

In a related aspect, the invention provides a computer program product comprising instructions for performing the method of the first aspect.

In a preferred embodiment of the sixth aspect, said device is provided on a liquid handling robot.

Relatedly, but in another aspect, the invention provides a liquid handling robot comprising a device of the sixth aspect.

In a seventh aspect the invention provides use of the device of the sixth aspect for performing the method of the first aspect.

In an eighth aspect, the present invention provides (a) an electrical conductor, preferably at least one coil, more preferably a Helmholtz coil, (b) at least one container having at least two openings, and (c) comprising or consisting of at least one magnetic material;
Elements (a), (b) and (c) are arranged such that (i) said at least one magnetic body is under the influence of the magnetic field generated by said coil in use, and (ii) in use A device is provided wherein liquid flowing through the tubular element is configured to contact the magnetic material.

Preferred embodiments of the method of the first aspect apply mutatis mutandis to the device of the eighth aspect, so long as they provide details of the electrical conductors, tubular elements and magnetic bodies.

For example, the device of the eighth aspect preferably includes a filter, mesh, membrane or sponge that retains the magnetic material, or the magnetic material, without significantly restricting movement of the magnetic material during use of the device. There may be wires, filaments, wires or springs connecting to the reactor.

A particularly preferred embodiment of the device includes a filter that is a molecular weight cutoff filter. Such filters retain all substances with molecular weights above the cutoff while allowing all molecules with molecular weights below the cutoff to pass. By passing the sample through such an equipped device in a continuous manner, fragments will pass through the filter if their size is below the cutoff and no further fragmentation into smaller fragments will occur. deaf.

This can be used to obtain a narrow distribution of fragment sizes.
In a ninth aspect, the invention provides use of the device of the eighth aspect for carrying out the method of any one of the second, third and fourth aspects and aspects related thereto. do.

In a tenth aspect, the present invention provides (a) a container and/or container array, (b) at least one magnetic body, preferably at least one magnetic body per container, (c) an electrical conductor, and (d) ) Optionally, providing a kit comprising or consisting of a manual having instructions for assembling the device of the sixth aspect and/or for performing the method of the first aspect.

It is understood that any container array can be used, including arrays established in the art such as microtiter plates. Arrays may be two-dimensional (e.g., 8 x 12, and the higher density known microtiter plates with, e.g., 384 and 1536 vessels) or one-dimensional (referred to as "strips"), e.g., 8 or 12 It can have individual containers.

Preferred embodiments of the method of the first aspect apply mutatis mutandis to the kit of the tenth aspect, so long as they provide details of the electrical conductor, container(s) and magnetic material. be.

In a related aspect, a kit is provided that includes a flow-through reactor, such as a tubular element, in place of the container or container array of (a).

These kits provide the components that allow the user to assemble the device of the invention, as further disclosed above. Kits also allow the user some flexibility, based on the components being provided separately, or to assemble tailored devices that meet the requirements of the desired application of the invention. to

In a preferred embodiment, said kit further comprises or consists of one or more buffers for preparing the previously defined solutions or suspensions.

In a further preferred embodiment, said kit further comprises or consists of one or more selected from proteins, enzymes, chemicals and catalysts.

First lane: 100 bp DNA ladder. Left: the yeast was only boiled. The DNA remained in the pocket and did not migrate to the agarose gel. Right: yeast treated with magnetic fragmentation. The signal in the pocket is reduced and a cloud of DNA fragments can be seen in the gel. Markers: PageRuler™ Plus pre-stained protein ladder. "Fragmentation": Proteins that have been processed by magnetic fragmentation. CA: carbonic anhydrase. BSA: bovine serum albumin. NIST: NIST human antibody. GP: glycogen phosphorylase. SDS-PAGE gel (Example 3) Agarose gel (Example 3) Cleavage of carbonic anhydrase (SDS-PAGE) SDS-PAGE of samples. Markers: Page Ruler Plus. A lower amount of intact carbonic anhydrase is observed for all samples #1-#12 when compared to sample 'Start'. The concentration of carbonic anhydrase decreased upon magnetic cleavage.

The examples illustrate the invention.
Example 1
Magnetic Fragmentation of DNA Materials Fresh S. cerevisiae containing 100 μg of protein. S. cerevisiae pellets were used for all experiments. Cylindrical 2mm x 2mm samarium cobalt magnets were used as magnets. Gels were stained with Midori Green (MG10) from Nippon Genetics. 1×TAE was prepared by dilution from 50×TAE (48.44 g Tris, 12.1 ml acetic acid, 3.7224 g EDTA in 200 ml H ₂ O). A 100 bp DNA ladder was used from Nippon Genetics (MWD100). A 6x DNA loading buffer was prepared by mixing 0.2 g cresol red (114472-5G) + 9 ml glycerol (G6279-500ML) + 21 ml _H2O . Agarose for gel preparation was purchased from Nippon Genetics (Ag01; #D00248).

Method Agarose gel was prepared by diluting 1.2 g of agarose in 60 ml of 1×TAE by boiling. After the solution had cooled, 5 μl of Midori Green was added and a small gel was poured. Sample preparations always contained 100 μg of yeast S. cerevisiae. It was prepared using S. cerevisiae cells. Cells were solubilized with 100 μl H ₂ O, pH 7 and boiled at 95° C. and 1000 rpm for 10 minutes. For magnetic fragmentation, a samarium-cobalt magnet was added and the cells were further incubated for 4 hours at a magnetic flux density of approximately 1 mT in a magnetic field with a frequency of 160 Hz. 2 μl of 6× loading dye was added to 10 μl of each sample and loaded onto the agarose gel. DNA ladder samples were loaded directly. The gel was run at 100V for approximately 1 hour.

Results See Figure 1 and its description.

Discussion Boiled but otherwise untreated yeast DNA is too large to migrate on an agarose gel as expected. Each lane appears free of DNA fragments, so yeast genomic DNA can be used to indicate DNA fragmentation. After treatment by magnetic fragmentation, dark shading is seen in the DNA gel indicating a range of fragments of various sizes ranging from 200 to 1500 bp in length.

Example 2
Magnetic Fragmentation of Proteins Materials Carbonic anhydrase (CA, sol.02.04.19), BSA (BioRad, 64124165), NIST mAb (14HBD-D-002, sol.16.02.19), and glycogen phosphorylase ( GP, sol.25.03.19) was used for fragmentation experiments. A PageRuler™ Plus pre-stained protein ladder, 10-250 kDa was used as a marker (Thermo Scientific™, 11832124). A cylindrical 3 mm×3 mm NdFeB magnet was used as the magnetic body. PAA gel was used for gel electrophoresis (BioRad Criterion Midi, 64316288). Samples were prepared for gel electrophoresis using 4x Laemmli sample buffer (BioRad, 64172364). 1× running buffer (25 mM Tris, 0.1% SDS, 192 mM glycine; BioRad, 20200415) was used during electrophoresis. Gels were stained with Coomassie Brilliant Blue R-250 (BioRad 64105295). 10% acetic acid (BioRad, 20200420) was used to destain the gel.

METHODS 100 μg of protein at a concentration of 1 μg/μl in ultrapure water was quantified in a single coil setup with 200 windings on a 1 cm inner diameter at a setting of 120 Hz at a flux density of approximately 1 mT. One Eppendorf 1.5 mL vessel was processed by magnetic fragmentation for 18 hours. 15 μl of solution containing 15 μg protein was mixed with 5 μl each of 4× loading dye (Laemmli buffer) and a total of 20 μl was loaded onto the PAA gel. SDS-PAGE was performed at 200V for 40 minutes. The gel was stained for 1 hour using Coomassie Brilliant Blue solution. Decolorization was performed in a microwave oven at 440 W using the decolorization solution, heated for 10 minutes, cooled for 1 minute and then rinsed with water. This cycle was repeated once.

Results See Figure 2.

DISCUSSION All proteins show a clearly reduced signal, with only a small signal remaining after treatment. This clearly indicates fragmentation and removal of pre-existing proteins. The generated fragments cannot be seen clearly as expected because the peptides are poorly stained in SDS-PAGE.

Example 3: Sections of different compound classes using the same parameters over a wide range of parameters Materials Neodymium permanent magnets with parylene coating were used (cylindrical; 2 mm x 2 mm). Silica beads (1 mm diameter) were used as additional non-magnetic materials. Reagents for SDS-PAGE analysis were provided by BioRad: Criterion Midi Gel was used for gel electrophoresis, protein loading dye was 4× Laemmli sample buffer, and staining buffer for the gel was It was Coomassie Brilliant Blue R-250. Running buffer was home-made with 25 mM Tris, 0.1% SDS, and 192 mM glycine, and destaining buffer was 10% acetic acid in deionized water. Thermo Fisher's Page Ruler Plus was used as a protein size marker.

Reagents for DNA gel analysis were provided by Nippon Genetics: agarose for agarose gels, Midori green as fluorescent dye for DNA. DNA loading buffer was made with 0.2 g cresol red and 9 ml glycerol. Running buffer was 1×TAE buffer diluted from home-made 50×TAE buffer (48.44 g Tris, 12.1 ml acetic acid, 3.7224 g EDTA in 200 ml H ₂ O). rice field. In-house yeast pellets (from 1 ml of S. cerevisiae solution in ultrapure water at OD ₆₀₀ =0.6) were used to demonstrate DNA fragmentation. Protein fragmentation was demonstrated using carbonic anhydrase (CA) from Sigma Aldrich.

A Helmholtz coil setup (440 mm circumference, 15 mm coil height, 70 windings, 6 layers, 12 windings per layer) was used to generate the external oscillating magnetic field. An online tone generator (https://onlinetonegenerator.com/) was used in combination with an amplifier (SMSL SA-50 2×50 W) to generate square waves at defined frequencies.

Methods Three frequencies were chosen to demonstrate fragmentation over a range of field strengths: 70 Hz, 100 Hz, 130 Hz.

DNA samples were processed using three standard yeast pellets per frequency setting. The pellet was resuspended in 100 μl ultrapure water, transferred to a 0.5 ml screw cap tube, and 1 magnet and 5 additional non-magnetic objects (see above) were added.

To process protein samples, three approaches of 25 μg CA each in 100 μl ultrapure water per frequency setting were performed and one magnet and five additional non-magnetic bodies were added.

To determine the resistance, a 2.6 m long cable connected in series with the Helmholtz coil was measured to be 0.5 ohms.

Before each experiment, the gaussmeter was calibrated and the magnetic field strength was measured at a fixed position of the coil. The sample was then placed in the coil and the process started. The voltage results were measured with an amplifier and the coil voltage was measured directly at the first and last winding of the coil.

All samples were processed for 2 hours.
Measurements were recorded and the current was calculated using the following formula (see Table 1):
I=V/R, where R=0.5 ohms After treatment, 20 μl of carbonic anhydrase sample (containing 5 μg of carbonic anhydrase) was mixed with 7 μl of 4x protein loading dye and shaken at 1000 rpm for 10 minutes. It was boiled at 95°C while stirring. Samples were then loaded on an SDS-PAGE gel together with an input sample (containing 5 μg of unfragmented carbonic anhydrase) prepared in the same manner. Gel electrophoresis was performed at 200V for 40 minutes. 20 μl of yeast sample was added with 4 μl of 6x DNA loading dye and loaded directly onto the agarose gel. For positive controls, S. The cerevisiae pellet (previous) was resuspended in water, boiled at 95° C. for 10 minutes with shaking at 1000 rpm, and sonicated (10 cycles, 30 seconds on/off, Diagenode Bioruptur). The negative control was a sample of untreated yeast DNA (20 μl yeast resuspended without fragmentation or sonication).

Preparation of agarose gel:
1.2 g of agarose was resuspended in 60 ml of 1×TAE buffer by stepwise boiling in a 440 W microwave oven until completely dissolved. After the solution had cooled, 5 μl of Midori green was added and a small gel with dimensions of 12.6 cm was poured. Electrophoresis was performed at 100V for 1 hour.

result

See also FIGS.
Samples were loaded in ascending order of frequency. CA0 is the starting sample, ie carbonic anhydrase before treatment with the described Helmholtz coil. This forms a distinct band between 25 kDa and 35 kDa. CA1-CA9 are treated carbonic anhydrase samples. Bands in the gel have disappeared.

Samples were loaded in ascending order of frequency. Yeast 0 is the starting sample, ie yeast before treatment with the described Helmholtz coil. One bright DNA band can be seen and adheres to the lane loading bag as expected. Y1-Y9 are treated yeast samples. Each lane shows faded bright DNA content distributed across the lane. DNA CO (control) is boiled and sonicated yeast. A faded and only slightly brighter DNA content can be seen in the lane.

Discussion The calculated flux densities were consistent with the measurements (see Table 3). Protein fragmentation occurred in the test range of frequencies from 70 Hz to 130 Hz (see Figure 3) or magnetic flux densities from 1.85 mT to 3.58 mT, respectively. Fragmentation of DNA occurred in the same range (see Figure 4).

It can therefore be concluded that DNA can be fragmented with the same settings as proteins over a wide range of parameters.

Examples 4-8: These examples demonstrate that the method of the present invention works well over a wide range of parameters that can be used to control the method.

Example 4: Magnetic Cleavage of a Single Protein at Different Time Intervals Materials Neodymium permanent magnets with parylene coating were used (cylindrical; 2 mm x 2 mm). Ceramic beads (1 mm diameter) were used for the additional non-magnetic material. Carbonic anhydrase (CA) was provided by Sigma Aldrich. PreOmics iST buffer was used for peptide cleanup (available as a kit). Ultrapure water (LC-MS grade) was provided by Thermo Fisher.

A Helmholtz coil setup was used to generate an external oscillating magnetic field. A customized power supply programmed to mimic a square wave was used.

Reagents for SDS-PAGE analysis were provided by BioRad: Criterion Midi Gel was used for gel electrophoresis, protein loading dye was 4× Laemmli sample buffer, and staining buffer for the gel was It was Coomassie Brilliant Blue R-250. Running buffer was home-made with 25 mM Tris, 0.1% SDS, and 192 mM glycine, and destaining buffer was 10% acetic acid in deionized water. Thermo Fisher's Page Ruler Plus was used as a protein size marker.

Method 50 μg of carbonic anhydrase in 100 μl of ultrapure water per sample are processed in 0.5 ml screw cap tubes with Helmholtz coils using 1 magnet and 5 additional non-magnetic bodies. (see Materials). Power was set to the following settings:
FreqGen: 250000; PWM period: 8191; PWM value: 4000 (corresponding to a frequency of 120Hz and a current of 1.4-1.6V).

For each incubation time, duplicate samples were made and split as follows:
- 10 minutes for samples #1 and #2 - 30 minutes for samples #3 and #4 - 60 minutes for samples #5 and #6 - 120 minutes for samples #7 and #8 After incubation , 10 μl of samples #3-#8 were loaded on an SDS gel and SDS-PAGE was performed at 200V for 40 minutes. A sample of 5 μg of carbonic anhydrase was also loaded on the gel to show the concentration before treatment, which was 0.5 μg/μl = 5 μg in 10 μl. See FIG.

The remaining solution was added along with 100 μl of STOP buffer (see PreOmics iST kit) and the following peptide cleanup was performed as described in the PreOmics iST protocol. The eluted peptide solution was dried in a 45° C. SpeedVac until all liquid was evaporated and the pellet dissolved in 16 μl LC-LOAD buffer (see PreOmics iST kit). 2 μl of dissolved peptide was loaded onto the LC-MS. Samples were analyzed on a ThermoFisher Scientific Easy n-LC 1200 system coupled with a Thermo LTQ Orbitrap XL. Peptides were separated on an in-house C18 column applying a 45 minute gradient and tandem mass spectrometry was performed using the DDA Top 10 method. MS/MS data were searched against the yeast database using MaxQuant software with default settings.

result

Discussion SDS-PAGE analysis shows remaining intact carbonic anhydrase after fragmentation compared to the input concentration of the carbonic anhydrase sample. Fragmentation was demonstrated to occur within different times, starting at 30 minutes and ending with complete fragmentation after 120 minutes.

Analysis of the peptide shows that magnetic cleavage occurred over the entire test time. Each period of incubation revealed peptides derived from carbonic anhydrase cleavage.

Example 5: Magnetic Cleavage of Proteins at Different Frequencies Materials Neodymium permanent magnets were used (cylindrical; 2 mm x 2 mm). PreOmics iST buffer was used for peptide cleanup (available as a kit). Ultrapure water (LC-MS grade) was provided by Thermo Fisher (pH 7). An in-house yeast pellet (from 1 ml of S. cerevisiae solution in ultrapure water with OD ₆₀₀ =0.6) containing 100 μg of protein was used as a sample with a complete proteome.

Single coil set-up (8 small coils in series, one coil containing one reaction vessel, one coil diameter 1.1 cm, coil diameter 2.0 cm) height) was used to generate an external oscillating magnetic field. An online tone generator (https://onlinetonegenerator.com/) was used in combination with an amplifier (SMSL SA-50 2×50 W) to generate square waves at defined frequencies.

Method Two standard yeast pellets were used per frequency setting. The pellet was resuspended in 100 μl ultrapure water in a 1.5 ml reaction vessel and one magnet was added per sample. Samples were incubated in the coil setup described above for 15 minutes at 12 V and various frequency settings.

After incubation, samples were purified according to standard iST protocols using PreOmics iST buffer.

After elution, samples were dried in a SpeedVac (45° C.) and resuspended in 40 μl LC-Load (PreOmics iST buffer).

2 μl of dissolved peptide was loaded onto the LC-MS. Samples were analyzed on a ThermoFisher Scientific Easy n-LC 1200 system coupled with a Thermo LTQ Orbitrap XL. Peptides were separated on an in-house C18 column applying a 45 minute gradient and tandem mass spectrometry was performed using the DDA Top 10 method. MS/MS data were searched against the yeast database using MaxQuant software with default settings.

result

Discussion Experiments have shown that proteins are cleaved at different frequencies. Particularly good results have been obtained for overlapping frequencies of 120 Hz and 1000 Hz.

For a single frequency, the amount of peptide revealed was found to be particularly high at frequencies around 240 Hz.

Example 6: Magnetic Cleavage of Proteins Using Different Number of Magnets Materials Permanent neodymium magnets were used (spherical, 2 mm diameter, nickel plated). PreOmics iST buffer was used for peptide cleanup (available as a kit). Ultrapure water (LC-MS grade) was provided by Thermo Fisher (pH 7). An in-house yeast pellet (from 1 ml S. cerevisiae solution at OD ₆₀₀ =0.6) was used as a sample with a complete proteome.

Single coil set-up (8 small coils in series, one coil containing one reaction vessel, one coil diameter 1.1 cm, coil diameter 2.0 cm) height) was used to generate an external oscillating magnetic field. An online tone generator (https://onlinetonegenerator.com/) was used in combination with an amplifier (SMSL SA-50 2×50 W) to generate square waves at defined frequencies.

Method Two standard yeast pellets were used per magnet quantity. The pellet was resuspended in 100 μl ultrapure water in a 1.5 ml reaction vessel and a defined amount of magnet was added to the sample.
Samples #1 and #2: 1 magnet samples #3 and #4: 2 magnet samples #5 and #6: 3 magnet samples #7 and #8: 4 magnet samples, maximum capacity output at 120 Hz for 1 hour with the above coil setup.

After incubation, samples were purified according to standard iST protocols using PreOmics iST buffer.

After elution, samples were dried in a SpeedVac (45° C.) and resuspended in 40 μl LC-Load (PreOmics iST buffer). 2 μl of dissolved peptide was loaded onto the LC-MS and the received chromatogram was analyzed using MaxQuant.

result

Discussion Experiments have shown that proteins are cleaved with different amounts of magnets. Particularly good results have been obtained with four magnets.

Example 7: Magnetic Cleavage of Proteins with Different Magnet Materials Materials Different permanent magnets were used (see Methods: Magnet with sample). PreOmics iST buffer was used for peptide cleanup (available as a kit). Ultrapure water (LC-MS grade) was provided by Thermo Fisher (pH 7). An in-house yeast pellet (from 1 ml S. cerevisiae solution at OD ₆₀₀ =0.6) was used as a sample with a complete proteome.

Single coil set-up (8 small coils in series, one coil containing one reaction vessel, one coil diameter 1.1 cm, coil diameter 2.0 cm) height) was used to generate an external oscillating magnetic field. An online tone generator (https://onlinetonegenerator.com/) was used in combination with an amplifier (SMSL SA-50 2×50 W) to generate square waves at defined frequencies.

Method Two standard yeast pellets were used per magnet type. The pellet was resuspended in 100 μl ultrapure water in a 1.5 ml reaction vessel and one magnet was added per sample. Samples were incubated at maximum volume at 120 Hz for 1 hour with the above coil setup and the magnet split as follows:
Sample #1+#2: 2×2 mm Neodymium disc N35
Sample #3+#4: 2×2 mm Neodymium disc N42
Sample #5+#6: 2 x 2 mm SaCo disc 0.11 kg pull Sample #7 + #8: 3 x 2 mm SaCo disc 0.19 kg pull Refined.

After elution, samples were dried in a SpeedVac (45° C.) and resuspended in 40 μl LC-Load (PreOmics iST buffer).

2 μl of dissolved peptide was loaded onto the LC-MS and the received chromatogram was analyzed using MaxQuant.

result

Discussion Experiments have shown that proteins are cleaved with different magnets. Particularly good results were obtained with a samarium-cobalt magnet, 3×2 mm, 0.19 kg pull.

Example 8: Magnetic Cleavage of Proteins with Different Magnet Materials, Shapes, Sizes and Coatings Materials Different permanent magnets were used (see Methods: Magnet by Sample). PreOmics iST buffer was used for peptide cleanup (available as a kit). Ultrapure water (LC-MS grade) was provided by Thermo Fisher (pH 7). An in-house yeast pellet (from 1 ml S. cerevisiae solution at OD ₆₀₀ =0.6) was used as a sample with a complete proteome.

Single coil set-up (8 small coils in series, one coil containing one reaction vessel, one coil diameter 1.1 cm, coil diameter 2.0 cm) height) was used to generate an external oscillating magnetic field. An online tone generator (https://onlinetonegenerator.com/) was used in combination with an amplifier (SMSL SA-50 2×50 W) to generate square waves at defined frequencies.

Method Two standard yeast pellets were used per magnet type. The pellet was resuspended in 100 μl ultrapure water in a 1.5 ml reaction vessel and one magnet was added per sample. Samples were incubated at maximum volume at 120 Hz for 1 hour with the above coil setup and the magnet split as follows:
Sample #1 + #2: Disk magnet φ5 mm, height 5 mm, ferrite sample #3 + #4: Spherical, φ5 mm, neodymium, gold-plated Sample #5 + #6: Disk magnet, φ3 mm, height 1 mm, neodymium, parylene coating sample # 7+#8: disk magnet, φ5 mm, height 5 mm, neodymium, Teflon (registered trademark) coated sample #9+#10: spherical φ3 mm, neodymium, chrome-plated sample #11+#12: disk magnet φ3 mm, height 1 mm, neodymium, Nickel-plated sample #13+#14: cuboid/cut stone 5×1.5×1 mm, neodymium, nickel-plated sample #15+#16: TiN-coated neodymium disc, φ3 mm, height 1 mm
Sample #17+#18: Spherical φ2 mm, neodymium, nickel-plated Sample #19+#20: Disc φ2 mm, height 2 mm, neodymium, nickel-plated Sample #21+#22: Disc φ3 mm, height 2 mm, neodymium, nickel-plated Sample #23+ #24: Disc φ 3 mm, Height 3 mm, Neodymium, Nickel Plated After incubation, samples were purified using PreOmics iST buffer following standard iST protocols.

After elution, samples were dried in a SpeedVac (45° C.) and resuspended in 40 μl LC-Load (PreOmics iST buffer).

2 μl of dissolved peptide was loaded onto the LC-MS and the received chromatogram was analyzed using MaxQuant.

result

Discussion Experiments have shown that proteins are cleaved with different magnets: different sizes, different materials, different shapes, and different coatings. Especially good results were obtained with a neodymium disc magnet, 3×1 mm, TiN coating.

Example 9: Magnetic Cleavage Using Different Corrugations Materials Neodymium permanent magnets with parylene coating were used (cylindrical; 2 mm x 2 mm). Silica beads of 1 mm were used as the non-magnetic material. A Helmholtz coil was used to generate an external oscillating magnetic field. An online tone generator (https://onlinetonegenerator.com/) was used to generate different waveforms at defined frequencies.

Materials for SDS-PAGE were purchased from Biorad (Criterion Midi Gel; 4x Laemmli sample buffer; Coomassie Brilliant Blue R-250) and Thermo Fisher (Page Ruler Plus). A home-made buffer was used to run the gel (25 mM Tris, 0.1% SDS, 192 mM glycine). The colored gel was destained with 10% diluted acetic acid.

Method 20 μg carbonic anhydrase per sample was treated in 50 μl ultrapure water (LC-MS grade, Fisher Scientific) in 0.5 ml screw cap tubes. One magnet and five non-magnetic bodies were added per sample. Samples were processed in triplicate for each waveform at 110 Hz for 90 minutes each in a container placed in a Helmholtz coil. Different waveforms were applied as follows:
Sample #1-Sample #3: Square wave function Sample #4-Sample #6: Sine function Sample #7-Sample #9: Sawtooth function
Sample #10-Sample #12: Trigonometric function After treatment, 20 μl of sample (equivalent to 8 μg of carbonic anhydrase initially used) was mixed with 7 μl of Laemmli sample buffer and heated at 95° C. with shaking at 1000 rpm for 10 minutes. Boiled and loaded on an SDS gel. Another sample corresponding to the starting amount of carbonic anhydrase originally used was prepared in the same manner. This so-called "starting" sample was also loaded onto the gel. SDS-PAGE was performed at 200V for 40 minutes.

Results See FIG.

Discussion This experiment demonstrates that magnetic cleavage of proteins can be performed with different waveforms.

Claims (15)

A method of cleaving at least one covalent bond in at least one molecule, said method comprising:
(a) colliding at least one magnetic substance with said at least one molecule; and/or (b) at least one non-magnetic particle and said at least one molecule by moving said at least one magnetic substance. A method comprising causing collisions with individual molecules. Said at least one magnetic body performs a fluctuating or oscillating motion, preferably said motion is caused by a fluctuating or oscillating magnetic field, preferably said magnetic field is generated by an electric current and/or an electromagnet A method according to claim 1. 3. The method of claim 1 or 2, wherein said collisions transfer a sufficient amount of energy to said molecule to cleave said at least one covalent bond. of the preceding claim, wherein said at least one molecule is in solution or suspension, and preferably said at least one molecule and said at least one magnetic material are located within a reactor. A method according to any one of paragraphs. 10. A process according to any one of the preceding claims, wherein the process is carried out batchwise, for example by using a reactor which is a vessel with a closed bottom. Said method is carried out in a continuous mode, for example said solution or suspension is preferably
(a) in a reactor having at least two openings,
is capable of flowing over the at least one magnetic material, and/or (b) motion of the magnetic material occurs while the solution or suspension is flowing;
5. The method of claim 4. The dimensions of said at least one magnetic body and said reactor are such that said at least one magnetic body is free or substantially free to move about its mean position, preferably in free motion or the substantially free motion is about or along at least two, at least three, at least four, at least five, or preferably all six axes of translational and rotational motion; 7. A method according to any one of claims 4 to 6, wherein yes, preferably said free or substantially free movement comprises at least translation along two axes. said at least one molecule is a macromolecule or chain molecule, said macromolecule or chain molecule comprising or consisting of at least two building blocks, preferably said building blocks are amino acids, 4. A method according to any one of the preceding claims, which is a nucleotide, ribonucleotide, saccharide, phosphate, lipid, fatty acid, sugar, glyceride, or a combination of any of the foregoing. contacting said at least one molecule with a further means for cleaving at least one covalent bond of said at least one molecule, said further means preferably being an enzyme, a chemical and/or a catalyst; A method according to any one of the preceding claims. (a) said solution or suspension is a liquid sample such as a buffer or body fluid, said molecule is a nucleic acid;
(b) said solution or suspension is drinking water or pool water and said molecule is a macromolecular contaminant;
(c) the solution or suspension is sewage, the molecule is a biomacromolecule;
(d) said solution or suspension is a food or beverage and said molecule is gluten;
(e) said solution or suspension comprises proteins, polypeptides and/or peptides, proteinaceous ingredients for growth media and/or proteinaceous ingredients for personal care products such as shampoos, conditioners or soaps, said the molecule is said or a protein, polypeptide and/or peptide;
(f) said solution or suspension comprises a macromolecular toxin and said molecule is said macromolecular toxin; or (g) said solution or suspension comprises undesirable enzymatic activity and said molecule is a protein that exhibits the enzymatic activity,
10. A method according to any one of claims 4-9. Use of magnetic materials and means to generate a varying or oscillating magnetic field to cleave at least one covalent bond in at least one molecule. (a) an electrical conductor, preferably at least one coil, more preferably a Helmholtz coil;
(b) a container and/or container array, e.g., a microtiter plate, and (c) at least one magnetic substance within said container, or at least one magnetic substance per container within said container array, or consists of these
a magnetic field generated by said at least one magnetic body in use by said conductor, preferably by implementing said conductor as a coil and by said container or said container array being positioned within said coil elements (a) and (b) are configured such that they are under the influence of
the dimensions of the magnetic body and the container are such that, in operation, the magnetic body is free to move along at least two axes of translation;
said magnetic body comprises or consists of a ferromagnetic or ferrimagnetic material;
device. A liquid handling robot comprising a device according to claim 12. (a) an electrical conductor, preferably at least one coil, more preferably a Helmholtz coil;
(b) at least one container having at least two openings; and (c) at least one magnetic material,
Elements (a), (b), and (c) are
(i) the at least one magnetic body is under the influence of the magnetic field generated by the coil in use; and (ii) in use the liquid flowing through the tubular element is in contact with the magnetic body. to the
is composed of
said magnetic body comprises or consists of a ferromagnetic or ferrimagnetic material;
device. (a) a container and/or container array;
(b) at least one magnetic body, preferably at least one magnetic body per container;
(c) an electrical conductor; and (d) optionally instructions for assembling the device of claim 14 and/or performing the method of any one of claims 1-10. including or consisting of a manual having
the dimensions of the magnetic body and the container are such that, in operation, the magnetic body is free to move along at least two axes of translation;
said magnetic body comprises or consists of a ferromagnetic or ferrimagnetic material;
kit.

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